Alphitobius diaperinus (Coleoptera: Tenebrionidae) Susceptibility to Cunila angustifolia Essential Oil Author(s): Geisa Percio Do Prado , Lenita Moura Stefani , Aleksandro Schafer Da Silva , Lisonéia Fiorentini Smaniotto , Flávio Roberto Mello Garcia , and Neusa Fernandes De Moura Source: Journal of Medical Entomology, 50(5):1040-1045. 2013. Published By: Entomological Society of America URL: http://www.bioone.org/doi/full/10.1603/ME12277
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VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS
Alphitobius diaperinus (Coleoptera: Tenebrionidae) Susceptibility to Cunila angustifolia Essential Oil GEISA PERCIO DO PRADO,1,2 LENITA MOURA STEFANI,1 ALEKSANDRO SCHAFER DA SILVA,1 LISONE´IA FIORENTINI SMANIOTTO,3 FLA´VIO ROBERTO MELLO GARCIA,4 5 AND NEUSA FERNANDES DE MOURA
J. Med. Entomol. 50(5): 1040Ð1045 (2013); DOI: http://dx.doi.org/10.1603/ME12277
ABSTRACT The lesser mealworm, Alphitobius diaperinus (Panzer), is an insect that lives in poultry houses, and high infestations may cause economic losses to producers. The control of this insect is usually done with insecticides; however, many of these chemicals have no effect on lesser mealworm. Therefore, control alternatives are needed. The aim of this study was to evaluate the inßuence of Cunila angustifolia (Benth) oil on larvae and adults of A. diaperinus. In vitro tests used larvae and adults of A. diaperinus distributed in petri dishes with 0, 1, 5, and 10% of oil in a single dose. In vivo tests were performed in poultry houses with Þve treatments: 0, 5, and 10% and chemical insecticide (cypermethrin) in a single application, and a group with 5% of oil applied twice 15 d apart. In vitro, oil bioactivity showed an efÞcacy of 100% both for larvae and adults, when tested at concentrations of 5 and 10%. A reduced number of larvae were observed using 1% of oil; however, it was not effective against adults as compared with the control group. In vivo, the oil effectiveness against lesser mealworm was veriÞed by larva and adult reduction in all concentrations compared with control (0%) throughout the experiments, with better efÞcacy when used at 5% with two applications. Therefore, we concluded that the oil of C. angustifolia has larvicidal and insecticidal effect against A. diaperinus larvae and adults, in vitro and in vivo. KEY WORDS Cunila angustifolia, essential oil, lesser mealworm
The poultry industry has made remarkable advances worldwide in recent decades. However, some challenges remain and among them are pest problems associated with Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), also known as the lesser mealworm. This insect has a worldwide distribution, causes major economic losses, and is a potential reservoir for enteric bacteria responsible for food illness worldwide (McAllister et al. 1994, 1996). The lesser mealworm is an omnivore that feeds on fecal material, spilled chicken feed, cracked eggs, chicken carcasses, house ßy maggots, and detritus (Axtell 1999). These insects are responsible for poor feed conversion because birds eat and digest the mealworm instead of a well-balanced feed. In addition, they may transmit pathogens to healthy birds, such as fungus (Aspergillus sp.), bacteria (Escherichia sp., Salmonella sp., Bacillus sp., and Streptococcus sp.), viruses, and parasites 1 Department Animal Science and Nursery, Universidade do Estado de Santa Catarina (UDESC), Chapeco´ , SC, Brazil 89815-630. 2 Corresponding author, e-mail: [email protected]. 3 Department EntomZoologia (Entomology), Universidade Federal do Parana´ (UFPR), Curitiba, PR, 81531-980 Brazil. 4 Department of Ecology, Zoology and Genetics, Lab of Insect Ecology, Universidade Federal de Pelotas (UFPEL), Pelotas, RS, 96010-900 Brazil. 5 Department of Chemical and Food School, Universidade Federal de Rio Grande (FURG), Rio Grande, RS, 95500-000 Brazil.
(Eimeria sp., Choanotaenia sp., and Raillietina sp.) (De Las Casas et al. 1972, ChernakiÐLeffer et al. 2002). Synthetic insecticides like cypermethrin and dichlorvos have low efÞcacy against the mealworm (ChernakiÐLeffer et al. 2011). Currently, there is great interest in using plant extracts as an alternative to chemical insecticides for pest control that are natural, easily obtained, rapidly degraded, with low cost, and without risk of meat residue (Prado 2007). Cunila angustifolia, also known as vassourinha, is a leafy shrub of ⬇1.5 m of height. The leaves of this plant are petiolate with leaf blade of 6 Ð 60 mm long and 1Ð15 mm wide, elliptic-lanceolate, obtuse, slightly toothed margins from half to three quarters of the leaf (Xifreda and Mallo 2001). The essential oil of C. angustifolia is rich in oxygenated compounds, with pulegone (Prado et al. 2008) that has proven insecticidal activity to primary active ingredient (Prado 2007). This study aims to investigate the efÞcacy of essential oil of C. angustifolia in the control of lesser mealworm (A. diaperinus) with in vitro and in vivo tests. Materials and Methods Oil Extraction. Leaves of C. angustifolia were collected in Chapeco´ City, Santa Catarina State, Brazil. Species identiÞcation (SMDB 9925) was performed,
0022-2585/13/1040Ð1045$04.00/0 䉷 2013 Entomological Society of America
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PRADO ET AL.: A. diaperinus SUSCEPTIBILITY TO C. angustifolia OIL
and samples were deposited in the Herbarium of the Federal University of Santa Maria (UFSM), Rio Grande do Sul, Brazil. After harvesting, leaves were subjected to steam distillation in Cleavenger-modiÞed apparatus for a period of 4 h. The essential oil extract was separated from the water using ethyl ether. The organic phase was treated with anhydrous sodium sulfate, Þltered, and the solvent was removed on a rotary evaporator. The residual oil was packaged in amber bottles and kept under refrigeration. The yield of essential oil extraction was 1.2%. Oil chemical composition was evaluated as previously published by Prado et al. (2008). For the in vitro and in vivo tests, the oil was diluted in Tween (1 v/v, i.e., 50% oil and 50% diluents) and subsequently in water at different concentrations. Lesser Mealworm. Samples of A. diaperinus were collected in poultry farms located in Chapeco´ , Santa Catarina, Brazil. This A. diaperinus infestation was natural, as the insect remained in the litter after the last batch of chickens was removed, although new litter was placed. The identiÞcation and separation of adults and larvae in the last stage (L8) of their life cycle were performed according to Chernaki and Almeida (2001a). In Vitro Test. For the in vitro test, petri dishesÑ10 cm in diameter and 2 cm in heightÑwere lined with paper Þlter with oil (100 l) at concentrations of 1, 5, and 10% diluted in water. To validate the test, a control group was also used, treated only with tween diluted in distilled water. Twenty-four hours after the start of testing, the paper Þlter was removed and pelleted commercial ration and cotton soaked in water were added to the plate for food and hydration of insects. This procedure was carried out every 3 d for a period of 30 d. Each petri dish contained 10 adults and 10 larvae (L8 stage). For each treatment, there were three simultaneous replicates in a completely randomized design. The experiment was conducted under a temperature of 25⬚C (⫾3⬚C) and relative humidity of 70% (⫾10%), with a photoperiod of 12:12 (L:D) h. Subsequently, for 30 d all live individuals were counted. In Vivo Test. At the hatchery, all birds were vaccinated against bursal infectious disease and MarekÕs disease. The in vivo test was conducted in an experimental poultry house, divided into 15 boxes (area of 4 m2 each), with new litter of pine shavings (40 chicks per box). They were supplied with feed, and received technical supervision from veterinarians. The essential oil was applied in different concentrations and application frequency, forming different groups: 5% (a single applicationÑ250 ml/m2), 5% (two applications with an interval of 15 dÑ250 ml/ m2), 10% (a single applicationÑ250 ml/m2), negative control (a single application of water and tween (vol: vol)Ñ250 ml/m2), and positive control (treated with chemical insecticideÑ cypermethrin 3 g/m2, diluted in water). The tests were performed with three repetitions for each treatment. In every box (2 by 2 m), Þve traps were placed under the feeder and drinker, in the
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center and at the entrance of the box, and in front of the window to capture larvae (all stages) and adult mealworms. The traps used are known as “Arends trap” described by Godinho and Alves (2009). The essential oil was applied in 1,000-ml solution of water at different concentrations in each treatment. Similarly to other groups, the same volume of water with cypermethrin was used in the positive control group. In all groups, application was made with manual applicator after litter placement, which corresponds to a day before placing 1-d-old chicks (day zero), at a density of 40 chicks per box. As mentioned above, the group treated with 5% had a second application 15 d after placement with the birds present. Subsequently, weekly counts (days 1, 7, 15, 21, 28, 35, 42, and 50) of the population of adult insects (dead and alive) and larvae (living and dead) in the traps were made. During the experiment, the temperature inside the poultry house was controlled according to the animal growth phase varying from 24 to 32⬚C, because according to the literature at temperatures below 16.5⬚C, the amount of insects is lower (Chernaki and Almeida 2001b). Animal behavior was observed during the entire experiment to identify possible oil intoxication. Statistical Analysis. Data from in vitro and in vivo tests were subjected to normality test (normal data), followed by analysis of variance (ANOVA) and Duncan test. These results were considered signiÞcant when P ⬍ 0.05. Results In Vitro. The results from the in vitro studies are shown in Fig. 1. The 1% oil concentration had no effect against adult stages of the mealworm, unlike what occurred with 5 and 10%, in which A. diaperinus adults were killed ⬍24 h after administration (Fig. 1A). When oil was tested on larvae, we observed the process to be dose-dependent. All three concentrations tested killed all the larvae, and the concentration of 5 and 10% were 100% effective in ⬍24 h (Fig. 1B). In Vivo. Larvae. Results from the in vivo test using C. angustifolia oil against mealworm larvae held in an experimental poultry house are presented in Table 1. SigniÞcantly fewer larvae were observed in all treatments with oil when compared with control for up to 21 d of the experiment. The number of larvae was not signiÞcantly different when 5 and 10% of oil was used after 28 d of the beginning of the study. Different results were observed when 5% of oil with two applications was used where signiÞcant lower number of larvae were captured, which shows improved insecticidal efÞcacy of this treatment (Table 1). At the end of the experiment, the number of larvae collected throughout the study was quantiÞed, that is, 13,760, 8,878, 2,705, 8,323, and 10 larvae per treatment at 0% (control), 5%, 5% in two application, 10%, and cypermethrin, respectively. Adults. The effect of C. angustifolia oil against adult mealworm in vivo is shown in Table 2. Treatments with oil at a 5% concentration in a single dose and with
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Fig. 1. In vitro dose-dependent effect of C. angustifolia oil on adult stages (A) and larvae (B) of A. diaperinus (n ⫽ 10) at 0 (control), 1, 5, and 10%. Results in the same column (day posttreatment) and within a circle are not statistically different among themselves in Duncan test (P ⬎ 0.05).
two applications signiÞcantly reduced the number of adult insects when compared with the control group for the duration of the experiment. At 10% concentration of the oil, it was observed a reduction of adult mealworms for up to day 28 compared with the controls. After day 28 (days 35, 42, and 50), we found no statistical difference from the control group (Table 2). At the end of the study, the number of adults A. diaperinus collected throughout the experiment was 1,576, 491, 354, 1,179, and 55 for 0% (control), 5%, 5% in two applications, 10%, and cypermethrin, respectively. Treatment Effect on Birds. No behavioral differences were observed soon after the chicks were housed on litter treated with oil nor on those treated with the chemical insecticide, so apparently the treatments were not toxic to the animals. The second administration of oil at 5% performed in the presence of birds 15 d after the Þrst dose showed no apparent changes indicative of clinical signs of intoxication. Discussion The control of the A. diaperineus is considered difÞcult, and done in most properties with the use of chemical insecticides such as cypermethrin, dichlorvos, trißumuron, cyßuthrin, among others (Salin et al. 2003, Japp et al. 2010, ChernakiÐLeffer et al. 2011). However, this method has disadvantages because it
cannot be used when the birds are in the poultry house. In the current study, we showed that C. angustifolia oil can be used as an insecticide against larvae and adult forms of A. diaperinus, which may represent a new strategy to control this pest, despite the fact that cypermethrin causes higher beetle mortality. Therefore, C. angustifolia oil could be used to control A. diaperinus when birds are in the aviary and cypermethrin used only for litter treatments when the poultry house is vacant. Researchers have suggested natural and effective alternatives for the control of lesser mealworms with the use of oils from Ocotea odorifera and Eucalyptus viminalis (JuniorÐPinto et al. 2010), Azadirachta indica and Cymbopogon winterianus (Azevedo et al. 2010, Marques 2010), and also by the use of pathogenic fungi such as Beauveria bassiana and Metarhizium anisopliae (Alves et al. 2004, Da Silva et al. 2006). C. angustifolia oil used in this study showed a higher concentration of not oxygenated monoterpenes as published previously by Prado et al. (2008). However, it is believed that the toxic action against A. diaperinus is because of the concentration of oxygenated terpenoids, such as the pulegone. It is known that the pulegone presents hepatotoxic effect, and in some in vivo studies with rodents, this terpene demonstrated toxicity (Gordon et al., 1982, ScientiÞc Committee on Food 2002, Simas et al., 2003). The metabolism of pulegone can lead to the formation of menthofuran,
Day 7
18.00a (⫾3.36) 5.60b (⫾1.5) 0.00c (⫾0.0) 9.10b (⫾2.05) 0.0c (⫾0.0)
Day 1
6.33a (⫾1.07)a 0.00b (⫾0.0) 0.00b (⫾0.0) 0.66b (⫾0.19) 0.0b (⫾0.0) 116.66a (⫾23.84) 18.33b (⫾2.21) 0.33d (⫾0.19) 4.66c (⫾0.19) 0.0d (⫾0.0)
Day 15 378.40a (⫾67.05) 86.56b (⫾16.02) 12.14c (⫾3.03) 9.33c (⫾0.83) 1.0d (⫾0.17)
Day 21 307.36a (⫾54.7) 456.60a (⫾99.07) 12.00b (⫾3.93) 296.23a (⫾64.2) 0.0c (⫾0.0)
Day 28
Day 35 1097.31a (⫾134.4) 937.54ab (⫾174.0) 147.66c (⫾34.4) 707.33b (⫾98.7) 0.0d (⫾0.0)
Average and SEs of the no. of larvae per treatment Day 42 723.00b (⫾119.3) 557.42bc (⫾35.4) 375.47c (⫾71.7) 1171.3a (⫾110.4) 2.33d (⫾0.53)
Day 50 1937.21a (⫾316.6) 896.94b (⫾69.96) 354.74c (⫾62.3) 464.17c (⫾81.3) 0.0d (⫾0.0)
Day 7
38.66a (⫾3.16) 15.33b (⫾4.01) 15.00b (⫾2.88) 11.00b (⫾2.08) 2.0c (⫾1.1)
Day 1
3.33a (⫾0.19)a 4.00a (⫾1.20) 3.00a (⫾0.56) 5.63a (⫾1.03) 8.63a (⫾2.52)
130.00a (⫾5.68) 21.33c (⫾5.29) 42.66bc (⫾6.86) 62.60b (⫾3.65) 5.0d (⫾1.16)
Day 21
32.66a (⫾0.84) 17.66b (⫾3.26) 4.60c (⫾1.26) 22.00b (⫾1.00) 0.66c (⫾0.38)
Day 28
53.30a (⫾9.71) 22.33b (⫾1.26) 12.66c (⫾2.0) 34.00abc (⫾6.69) 0.0d (⫾0.0)
Day 35
Average and standard errors of the no. of mealworms per treatment 60.33a (⫾6.50) 16.66b (⫾1.34) 5.00c (⫾1.15) 7.66c (⫾0.83) 0.33d (⫾0.18)
Day 15
Quantitation of adult mealworms was performed on days 1, 7, 15, 28, 35, 42, and 50 after treatment. a Mean with the same letters in the same column do not differ statistically among themselves, with the probability of 5% in the Duncan test.
Control Oil 5% Oil 5%/2⫻ Oil 10% Cypermethrin
Treatment
109.33a (⫾14.34) 16.66b (⫾4.22) 24.33b (⫾1.50) 134.66a (⫾25.16) 0.33d (⫾0.18)
Day 42
97.06a (⫾14.56) 49.66b (⫾5.93) 10.63c (⫾2.77) 115.33a (⫾16.7) 0.0d (⫾0.0)
Day 50
Table 2. Number of A. diaperinus adults captured in each treatment of C. angustifolia oil at concentrations of 0% (negative control), 5%, 5% with two applications interval of 15 d (5% oil/2ⴛ), 10%, and chemical insecticide— cypermethrin (positive control)
QuantiÞcation of larvae was performed on days 1, 7, 15, 28, 35, 42, and 50 after treatment. a Mean with the same letters in the same column do not differ statistically among themselves, with the probability of 5% in the Duncan test.
Control Oil 5% Oil 5% in 2⫻ Oil 10% Cypermethrin
Treatment
Table 1. Number of A. diaperinus larvae captured in each C. angustifolia oil treatment at concentrations of 0% (negative control), 5%, 5% with two applications 15 d apart, 10%, and chemical insecticide— cypermethrin (positive control)
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which has a high toxicity (Gordon et al. 1982, 1987). However, this study did not investigated whether the essential oil caused some toxicity to birds, but apparently no signs of intoxication were observed after administration of the treatment. In a study with pulegone, an insecticide effect was observed against Tyrophagus putrescentiae (Sa´nchezÐ Ramos and Castan˜ era 2000), Sitophilus oryzae, Tribolium castaneum, Oryzaephilus surinamensis, Musca domestica, and Blattella germanica (Lee et al. 2003). Toxic effect was also observed for the ␣-pinene and camphene (not oxygenated monoterpenes) against Rhyzophagos dispar, Formica rupa, and Tenebrio molito (Viegas 2003). Therefore, C. angustifolia oil that contains pulegone showed good insecticidal effect on larvae and adults of A. diaperinus in vitro. This effect is probably because of the inhibition of acetylcholinesterase, which has been observed by other researchers that evaluated the action of 1,2-epoxy-pulegone on Lippia stoechadoifolia (Godfrey 1994). Our results show that C. angustifolia essential oil had insecticidal effect on adults and larvae of A. diaperinus, with the exception of the 1% concentration in vitro against adults. In vivo tests conÞrmed this effect because a reduction on the number of larvae and adults of lesser mealworm was observed. The treatment with 5% of oil with two applications showed better efÞcacy after day 21 (larvae) and 28 (adults) compared with other treatments and control group. This result can be explained by the fact that the second application may cause a prolonged residual effect of oil in the litter in 15 d, and it might be a better treatment option. In this study, it was found that treatment with C. angustifolia essential oil at all concentrations tested was able to reduce the number of A. diaperinus throughout the experiment when compared with control. As a result, we conclude that oil C. angustifolia has insecticidal action on lesser mealworm, and might be used as an alternative treatment to control this pest.
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Simas, N. K., E. C. Lima, S. R. Conceic¸ a˜ o, R. M. Kuster, and A. M. Oliveira–Filho. 2003. Produtos naturais para o controle da transmissa˜o da dengue: atividade larvicida de Myroxylon balsamum (o´ leo vermelho) e de terpeno´ ides e fenilpropano´ ides. Quõ´m. Nova 27: 46 Ð 49. Viegas, C., Jr. 2003. Terpenos com atividade inseticida: uma alternativa para o controle quõ´mico de insetos. Quõ´m. Nova 26: 390 Ð 400. Xifreda, C. C., and A. C. Mallo. 2001. Citas nuevas o crõ´ticas para la ßora argentina III: Cunila angustifolia (Lamiaceae: Mentheae). Darwiniana 39: 175Ð178. Received 17 December 2012; accepted 27 May 2013.
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VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS
Alphitobius diaperinus (Coleoptera: Tenebrionidae) Susceptibility to Cunila angustifolia Essential Oil GEISA PERCIO DO PRADO,1,2 LENITA MOURA STEFANI,1 ALEKSANDRO SCHAFER DA SILVA,1 LISONE´IA FIORENTINI SMANIOTTO,3 FLA´VIO ROBERTO MELLO GARCIA,4 5 AND NEUSA FERNANDES DE MOURA
J. Med. Entomol. 50(5): 1040Ð1045 (2013); DOI: http://dx.doi.org/10.1603/ME12277
ABSTRACT The lesser mealworm, Alphitobius diaperinus (Panzer), is an insect that lives in poultry houses, and high infestations may cause economic losses to producers. The control of this insect is usually done with insecticides; however, many of these chemicals have no effect on lesser mealworm. Therefore, control alternatives are needed. The aim of this study was to evaluate the inßuence of Cunila angustifolia (Benth) oil on larvae and adults of A. diaperinus. In vitro tests used larvae and adults of A. diaperinus distributed in petri dishes with 0, 1, 5, and 10% of oil in a single dose. In vivo tests were performed in poultry houses with Þve treatments: 0, 5, and 10% and chemical insecticide (cypermethrin) in a single application, and a group with 5% of oil applied twice 15 d apart. In vitro, oil bioactivity showed an efÞcacy of 100% both for larvae and adults, when tested at concentrations of 5 and 10%. A reduced number of larvae were observed using 1% of oil; however, it was not effective against adults as compared with the control group. In vivo, the oil effectiveness against lesser mealworm was veriÞed by larva and adult reduction in all concentrations compared with control (0%) throughout the experiments, with better efÞcacy when used at 5% with two applications. Therefore, we concluded that the oil of C. angustifolia has larvicidal and insecticidal effect against A. diaperinus larvae and adults, in vitro and in vivo. KEY WORDS Cunila angustifolia, essential oil, lesser mealworm
The poultry industry has made remarkable advances worldwide in recent decades. However, some challenges remain and among them are pest problems associated with Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae), also known as the lesser mealworm. This insect has a worldwide distribution, causes major economic losses, and is a potential reservoir for enteric bacteria responsible for food illness worldwide (McAllister et al. 1994, 1996). The lesser mealworm is an omnivore that feeds on fecal material, spilled chicken feed, cracked eggs, chicken carcasses, house ßy maggots, and detritus (Axtell 1999). These insects are responsible for poor feed conversion because birds eat and digest the mealworm instead of a well-balanced feed. In addition, they may transmit pathogens to healthy birds, such as fungus (Aspergillus sp.), bacteria (Escherichia sp., Salmonella sp., Bacillus sp., and Streptococcus sp.), viruses, and parasites 1 Department Animal Science and Nursery, Universidade do Estado de Santa Catarina (UDESC), Chapeco´ , SC, Brazil 89815-630. 2 Corresponding author, e-mail: [email protected]. 3 Department EntomZoologia (Entomology), Universidade Federal do Parana´ (UFPR), Curitiba, PR, 81531-980 Brazil. 4 Department of Ecology, Zoology and Genetics, Lab of Insect Ecology, Universidade Federal de Pelotas (UFPEL), Pelotas, RS, 96010-900 Brazil. 5 Department of Chemical and Food School, Universidade Federal de Rio Grande (FURG), Rio Grande, RS, 95500-000 Brazil.
(Eimeria sp., Choanotaenia sp., and Raillietina sp.) (De Las Casas et al. 1972, ChernakiÐLeffer et al. 2002). Synthetic insecticides like cypermethrin and dichlorvos have low efÞcacy against the mealworm (ChernakiÐLeffer et al. 2011). Currently, there is great interest in using plant extracts as an alternative to chemical insecticides for pest control that are natural, easily obtained, rapidly degraded, with low cost, and without risk of meat residue (Prado 2007). Cunila angustifolia, also known as vassourinha, is a leafy shrub of ⬇1.5 m of height. The leaves of this plant are petiolate with leaf blade of 6 Ð 60 mm long and 1Ð15 mm wide, elliptic-lanceolate, obtuse, slightly toothed margins from half to three quarters of the leaf (Xifreda and Mallo 2001). The essential oil of C. angustifolia is rich in oxygenated compounds, with pulegone (Prado et al. 2008) that has proven insecticidal activity to primary active ingredient (Prado 2007). This study aims to investigate the efÞcacy of essential oil of C. angustifolia in the control of lesser mealworm (A. diaperinus) with in vitro and in vivo tests. Materials and Methods Oil Extraction. Leaves of C. angustifolia were collected in Chapeco´ City, Santa Catarina State, Brazil. Species identiÞcation (SMDB 9925) was performed,
0022-2585/13/1040Ð1045$04.00/0 䉷 2013 Entomological Society of America
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PRADO ET AL.: A. diaperinus SUSCEPTIBILITY TO C. angustifolia OIL
and samples were deposited in the Herbarium of the Federal University of Santa Maria (UFSM), Rio Grande do Sul, Brazil. After harvesting, leaves were subjected to steam distillation in Cleavenger-modiÞed apparatus for a period of 4 h. The essential oil extract was separated from the water using ethyl ether. The organic phase was treated with anhydrous sodium sulfate, Þltered, and the solvent was removed on a rotary evaporator. The residual oil was packaged in amber bottles and kept under refrigeration. The yield of essential oil extraction was 1.2%. Oil chemical composition was evaluated as previously published by Prado et al. (2008). For the in vitro and in vivo tests, the oil was diluted in Tween (1 v/v, i.e., 50% oil and 50% diluents) and subsequently in water at different concentrations. Lesser Mealworm. Samples of A. diaperinus were collected in poultry farms located in Chapeco´ , Santa Catarina, Brazil. This A. diaperinus infestation was natural, as the insect remained in the litter after the last batch of chickens was removed, although new litter was placed. The identiÞcation and separation of adults and larvae in the last stage (L8) of their life cycle were performed according to Chernaki and Almeida (2001a). In Vitro Test. For the in vitro test, petri dishesÑ10 cm in diameter and 2 cm in heightÑwere lined with paper Þlter with oil (100 l) at concentrations of 1, 5, and 10% diluted in water. To validate the test, a control group was also used, treated only with tween diluted in distilled water. Twenty-four hours after the start of testing, the paper Þlter was removed and pelleted commercial ration and cotton soaked in water were added to the plate for food and hydration of insects. This procedure was carried out every 3 d for a period of 30 d. Each petri dish contained 10 adults and 10 larvae (L8 stage). For each treatment, there were three simultaneous replicates in a completely randomized design. The experiment was conducted under a temperature of 25⬚C (⫾3⬚C) and relative humidity of 70% (⫾10%), with a photoperiod of 12:12 (L:D) h. Subsequently, for 30 d all live individuals were counted. In Vivo Test. At the hatchery, all birds were vaccinated against bursal infectious disease and MarekÕs disease. The in vivo test was conducted in an experimental poultry house, divided into 15 boxes (area of 4 m2 each), with new litter of pine shavings (40 chicks per box). They were supplied with feed, and received technical supervision from veterinarians. The essential oil was applied in different concentrations and application frequency, forming different groups: 5% (a single applicationÑ250 ml/m2), 5% (two applications with an interval of 15 dÑ250 ml/ m2), 10% (a single applicationÑ250 ml/m2), negative control (a single application of water and tween (vol: vol)Ñ250 ml/m2), and positive control (treated with chemical insecticideÑ cypermethrin 3 g/m2, diluted in water). The tests were performed with three repetitions for each treatment. In every box (2 by 2 m), Þve traps were placed under the feeder and drinker, in the
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center and at the entrance of the box, and in front of the window to capture larvae (all stages) and adult mealworms. The traps used are known as “Arends trap” described by Godinho and Alves (2009). The essential oil was applied in 1,000-ml solution of water at different concentrations in each treatment. Similarly to other groups, the same volume of water with cypermethrin was used in the positive control group. In all groups, application was made with manual applicator after litter placement, which corresponds to a day before placing 1-d-old chicks (day zero), at a density of 40 chicks per box. As mentioned above, the group treated with 5% had a second application 15 d after placement with the birds present. Subsequently, weekly counts (days 1, 7, 15, 21, 28, 35, 42, and 50) of the population of adult insects (dead and alive) and larvae (living and dead) in the traps were made. During the experiment, the temperature inside the poultry house was controlled according to the animal growth phase varying from 24 to 32⬚C, because according to the literature at temperatures below 16.5⬚C, the amount of insects is lower (Chernaki and Almeida 2001b). Animal behavior was observed during the entire experiment to identify possible oil intoxication. Statistical Analysis. Data from in vitro and in vivo tests were subjected to normality test (normal data), followed by analysis of variance (ANOVA) and Duncan test. These results were considered signiÞcant when P ⬍ 0.05. Results In Vitro. The results from the in vitro studies are shown in Fig. 1. The 1% oil concentration had no effect against adult stages of the mealworm, unlike what occurred with 5 and 10%, in which A. diaperinus adults were killed ⬍24 h after administration (Fig. 1A). When oil was tested on larvae, we observed the process to be dose-dependent. All three concentrations tested killed all the larvae, and the concentration of 5 and 10% were 100% effective in ⬍24 h (Fig. 1B). In Vivo. Larvae. Results from the in vivo test using C. angustifolia oil against mealworm larvae held in an experimental poultry house are presented in Table 1. SigniÞcantly fewer larvae were observed in all treatments with oil when compared with control for up to 21 d of the experiment. The number of larvae was not signiÞcantly different when 5 and 10% of oil was used after 28 d of the beginning of the study. Different results were observed when 5% of oil with two applications was used where signiÞcant lower number of larvae were captured, which shows improved insecticidal efÞcacy of this treatment (Table 1). At the end of the experiment, the number of larvae collected throughout the study was quantiÞed, that is, 13,760, 8,878, 2,705, 8,323, and 10 larvae per treatment at 0% (control), 5%, 5% in two application, 10%, and cypermethrin, respectively. Adults. The effect of C. angustifolia oil against adult mealworm in vivo is shown in Table 2. Treatments with oil at a 5% concentration in a single dose and with
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Fig. 1. In vitro dose-dependent effect of C. angustifolia oil on adult stages (A) and larvae (B) of A. diaperinus (n ⫽ 10) at 0 (control), 1, 5, and 10%. Results in the same column (day posttreatment) and within a circle are not statistically different among themselves in Duncan test (P ⬎ 0.05).
two applications signiÞcantly reduced the number of adult insects when compared with the control group for the duration of the experiment. At 10% concentration of the oil, it was observed a reduction of adult mealworms for up to day 28 compared with the controls. After day 28 (days 35, 42, and 50), we found no statistical difference from the control group (Table 2). At the end of the study, the number of adults A. diaperinus collected throughout the experiment was 1,576, 491, 354, 1,179, and 55 for 0% (control), 5%, 5% in two applications, 10%, and cypermethrin, respectively. Treatment Effect on Birds. No behavioral differences were observed soon after the chicks were housed on litter treated with oil nor on those treated with the chemical insecticide, so apparently the treatments were not toxic to the animals. The second administration of oil at 5% performed in the presence of birds 15 d after the Þrst dose showed no apparent changes indicative of clinical signs of intoxication. Discussion The control of the A. diaperineus is considered difÞcult, and done in most properties with the use of chemical insecticides such as cypermethrin, dichlorvos, trißumuron, cyßuthrin, among others (Salin et al. 2003, Japp et al. 2010, ChernakiÐLeffer et al. 2011). However, this method has disadvantages because it
cannot be used when the birds are in the poultry house. In the current study, we showed that C. angustifolia oil can be used as an insecticide against larvae and adult forms of A. diaperinus, which may represent a new strategy to control this pest, despite the fact that cypermethrin causes higher beetle mortality. Therefore, C. angustifolia oil could be used to control A. diaperinus when birds are in the aviary and cypermethrin used only for litter treatments when the poultry house is vacant. Researchers have suggested natural and effective alternatives for the control of lesser mealworms with the use of oils from Ocotea odorifera and Eucalyptus viminalis (JuniorÐPinto et al. 2010), Azadirachta indica and Cymbopogon winterianus (Azevedo et al. 2010, Marques 2010), and also by the use of pathogenic fungi such as Beauveria bassiana and Metarhizium anisopliae (Alves et al. 2004, Da Silva et al. 2006). C. angustifolia oil used in this study showed a higher concentration of not oxygenated monoterpenes as published previously by Prado et al. (2008). However, it is believed that the toxic action against A. diaperinus is because of the concentration of oxygenated terpenoids, such as the pulegone. It is known that the pulegone presents hepatotoxic effect, and in some in vivo studies with rodents, this terpene demonstrated toxicity (Gordon et al., 1982, ScientiÞc Committee on Food 2002, Simas et al., 2003). The metabolism of pulegone can lead to the formation of menthofuran,
Day 7
18.00a (⫾3.36) 5.60b (⫾1.5) 0.00c (⫾0.0) 9.10b (⫾2.05) 0.0c (⫾0.0)
Day 1
6.33a (⫾1.07)a 0.00b (⫾0.0) 0.00b (⫾0.0) 0.66b (⫾0.19) 0.0b (⫾0.0) 116.66a (⫾23.84) 18.33b (⫾2.21) 0.33d (⫾0.19) 4.66c (⫾0.19) 0.0d (⫾0.0)
Day 15 378.40a (⫾67.05) 86.56b (⫾16.02) 12.14c (⫾3.03) 9.33c (⫾0.83) 1.0d (⫾0.17)
Day 21 307.36a (⫾54.7) 456.60a (⫾99.07) 12.00b (⫾3.93) 296.23a (⫾64.2) 0.0c (⫾0.0)
Day 28
Day 35 1097.31a (⫾134.4) 937.54ab (⫾174.0) 147.66c (⫾34.4) 707.33b (⫾98.7) 0.0d (⫾0.0)
Average and SEs of the no. of larvae per treatment Day 42 723.00b (⫾119.3) 557.42bc (⫾35.4) 375.47c (⫾71.7) 1171.3a (⫾110.4) 2.33d (⫾0.53)
Day 50 1937.21a (⫾316.6) 896.94b (⫾69.96) 354.74c (⫾62.3) 464.17c (⫾81.3) 0.0d (⫾0.0)
Day 7
38.66a (⫾3.16) 15.33b (⫾4.01) 15.00b (⫾2.88) 11.00b (⫾2.08) 2.0c (⫾1.1)
Day 1
3.33a (⫾0.19)a 4.00a (⫾1.20) 3.00a (⫾0.56) 5.63a (⫾1.03) 8.63a (⫾2.52)
130.00a (⫾5.68) 21.33c (⫾5.29) 42.66bc (⫾6.86) 62.60b (⫾3.65) 5.0d (⫾1.16)
Day 21
32.66a (⫾0.84) 17.66b (⫾3.26) 4.60c (⫾1.26) 22.00b (⫾1.00) 0.66c (⫾0.38)
Day 28
53.30a (⫾9.71) 22.33b (⫾1.26) 12.66c (⫾2.0) 34.00abc (⫾6.69) 0.0d (⫾0.0)
Day 35
Average and standard errors of the no. of mealworms per treatment 60.33a (⫾6.50) 16.66b (⫾1.34) 5.00c (⫾1.15) 7.66c (⫾0.83) 0.33d (⫾0.18)
Day 15
Quantitation of adult mealworms was performed on days 1, 7, 15, 28, 35, 42, and 50 after treatment. a Mean with the same letters in the same column do not differ statistically among themselves, with the probability of 5% in the Duncan test.
Control Oil 5% Oil 5%/2⫻ Oil 10% Cypermethrin
Treatment
109.33a (⫾14.34) 16.66b (⫾4.22) 24.33b (⫾1.50) 134.66a (⫾25.16) 0.33d (⫾0.18)
Day 42
97.06a (⫾14.56) 49.66b (⫾5.93) 10.63c (⫾2.77) 115.33a (⫾16.7) 0.0d (⫾0.0)
Day 50
Table 2. Number of A. diaperinus adults captured in each treatment of C. angustifolia oil at concentrations of 0% (negative control), 5%, 5% with two applications interval of 15 d (5% oil/2ⴛ), 10%, and chemical insecticide— cypermethrin (positive control)
QuantiÞcation of larvae was performed on days 1, 7, 15, 28, 35, 42, and 50 after treatment. a Mean with the same letters in the same column do not differ statistically among themselves, with the probability of 5% in the Duncan test.
Control Oil 5% Oil 5% in 2⫻ Oil 10% Cypermethrin
Treatment
Table 1. Number of A. diaperinus larvae captured in each C. angustifolia oil treatment at concentrations of 0% (negative control), 5%, 5% with two applications 15 d apart, 10%, and chemical insecticide— cypermethrin (positive control)
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which has a high toxicity (Gordon et al. 1982, 1987). However, this study did not investigated whether the essential oil caused some toxicity to birds, but apparently no signs of intoxication were observed after administration of the treatment. In a study with pulegone, an insecticide effect was observed against Tyrophagus putrescentiae (Sa´nchezÐ Ramos and Castan˜ era 2000), Sitophilus oryzae, Tribolium castaneum, Oryzaephilus surinamensis, Musca domestica, and Blattella germanica (Lee et al. 2003). Toxic effect was also observed for the ␣-pinene and camphene (not oxygenated monoterpenes) against Rhyzophagos dispar, Formica rupa, and Tenebrio molito (Viegas 2003). Therefore, C. angustifolia oil that contains pulegone showed good insecticidal effect on larvae and adults of A. diaperinus in vitro. This effect is probably because of the inhibition of acetylcholinesterase, which has been observed by other researchers that evaluated the action of 1,2-epoxy-pulegone on Lippia stoechadoifolia (Godfrey 1994). Our results show that C. angustifolia essential oil had insecticidal effect on adults and larvae of A. diaperinus, with the exception of the 1% concentration in vitro against adults. In vivo tests conÞrmed this effect because a reduction on the number of larvae and adults of lesser mealworm was observed. The treatment with 5% of oil with two applications showed better efÞcacy after day 21 (larvae) and 28 (adults) compared with other treatments and control group. This result can be explained by the fact that the second application may cause a prolonged residual effect of oil in the litter in 15 d, and it might be a better treatment option. In this study, it was found that treatment with C. angustifolia essential oil at all concentrations tested was able to reduce the number of A. diaperinus throughout the experiment when compared with control. As a result, we conclude that oil C. angustifolia has insecticidal action on lesser mealworm, and might be used as an alternative treatment to control this pest.
References Cited Alves, L.F.A., V. S. Alves, D. F. Brassan, P.M.O. Neves, and S. B. Alves. 2004. Ocorreˆ ncia de Metarhizium anisopliae (Metsch) Sorok em adultos de cascudinho (Alphitobius diaperinus) (Panzer) (Coleoptero: Tenebrionidae) em avia´rio comercial em Cascavel, PR. Neotrop. Entomol. 33: 793Ð795. Axtell, R. C. 1999. Poultry integrated pest management: status and future. Integr. Pest Manag. Rev. 4: 53Ð73. Azevedo, A.I.B., A. S. Lira, L. C. Cunha, F.A.C. Almeida, and R. P. Almeida. 2010. Bioatividade do o´ leo de nim sobre Alphitobius diaperinus (Coleoptera: Tenebrionidae) em sementes de amendoim. Rev. Bras. Eng. Agric. Ambient 14: 309 Ð313. Chernaki, A. M., and L. M. Almeida. 2001a. Morfologia dos esta´gios imaturos e do adulto de Alphitobius diaperinus (Panzer) (Coleoptera, Tenebrionidae). Rev. Bras. Zool. 18: 351Ð363. Chernaki, A. M., and L. M. Almeida. 2001b. Exigeˆncias te´rmicas, perõ´odo de desenvolvimento e sobreviveˆncia de imaturos de Alphitobius diaperinus (Panzer) (Coleoptera, Tenebrionidae). Neotrop. Entomol. 30: 365Ð368.
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Salin, C., Y. R. Delettre, and P. Vernon. 2003. Controlling the mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae) in broiler and turkey houses: Þeld trials with a combined insecticide treatment: insect growth regulator and pyrethroid. J. Econ. Entomol. 96: 126 Ð130. Sa´ nchez–Ramos, I., and P. Castan˜ era. 2000. Acaracidal activity of natural monoterpenes on Tyrophagus putrescentiae (Schrank), a mite of stored food. J. Stored Prod. Res. 37: 93Ð101. Scientific Committee on Food. 2002. Opinion of the scientiÞc committee on food on pulegone and menthofuran. Bruxelles/Brussel, Belgium. (http://ec.europa.eu/food/ fs/sc/scf/out133_en.pdf).
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Simas, N. K., E. C. Lima, S. R. Conceic¸ a˜ o, R. M. Kuster, and A. M. Oliveira–Filho. 2003. Produtos naturais para o controle da transmissa˜o da dengue: atividade larvicida de Myroxylon balsamum (o´ leo vermelho) e de terpeno´ ides e fenilpropano´ ides. Quõ´m. Nova 27: 46 Ð 49. Viegas, C., Jr. 2003. Terpenos com atividade inseticida: uma alternativa para o controle quõ´mico de insetos. Quõ´m. Nova 26: 390 Ð 400. Xifreda, C. C., and A. C. Mallo. 2001. Citas nuevas o crõ´ticas para la ßora argentina III: Cunila angustifolia (Lamiaceae: Mentheae). Darwiniana 39: 175Ð178. Received 17 December 2012; accepted 27 May 2013.
